Collagens, the most abundant proteins in connective tissues of higher eukaryotes, play important structural roles in many tissues and organs. At least 13 types of collagen have been described, which, due to their multisubunit structure, represent the products of at least 23 genes. Most of the collagen genes are developmentally regulated, such that a given tissue or cell type expresses only a subset of the collagen genes. Type I collagen, which this proposal will discuss, has been demonstrated in the chicken embryo as early as 4.5 days of development, primarily in presumptive dermis, somatic mesoderm, and the dermatomal myotomal plate. At later times in embryonic development, and in the adult, type I collagen is found in almost all connective tissues, but is generally excluded from cartilage. Surprisingly, the mRNAs encoding type I collagen have been observed both early in embryonic development (about day 1 of development) and in cartilage. The function of these type I collagen mRNAs early in embryogenesis and in cartilage, in the absence of detectable type I collagen protein production, is unknown. However, the molecular mechanism that appears to prevent production of alpha2(I) collagen in early embryos and in cartilage has been identified. A previously undescribed promoter and exon (exon A) have been found within intron 2 of the chick alpha2(I) collagen gene. Transcripts initiating at this promoter contain exon A instead of exons 1 and 2, and do not contain exon 7; there may be differences in other regions of the mRNA as well. Early in embryonic development and in cartilage transcription of the alpha2(I) collagen gene initiates almost exclusively at this newly identified promoter. Exon A contains an AUG initiating an open reading frame that is out of frame with the collagen coding sequence; thus the embryonic/cartilage alpha2(I) collagen mRNA apparently cannot encode alpha2(I) collagen. However, the mRNA is found in the cytoplasm and is associated with an average of three puromycin-releasable ribosomes (12). These results indicate that the mRNA is actively translated, but suggest that open reading frame may be relatively small. An antiserum prepared against the first 22 amino acids of the predicted open reading frame recognizes 35 and 33 kd proteins present in nuclei of early embryos and chondrocytes, but not in nuclei of other types of connective tissue cells. This application proposes experiments to identify the molecular mechanisms dictating use of the embryonic/cartilage promoter early in embryogenesis and in cartilage, and the use of the bone/tendon promoter in all other connective tissues. It also proposes experiments to determine the structure of the embryonic/cartilage form of the alpha2(I) collagen mRNA and to identify and characterize its protein product. The information provided by these experiments will give us increased insight into the developmentally regulated expression of this important gene family. In particular, they will provide increased understanding of the mechanisms by which production of type I collagen is restricted to the appropriate tissues and stages of embryonic development.